The mammalian nervous system has no mechanisms to replace lost neurons with the exception of some regions where neurogenesis continues throughout life (Kaplan et al., Science, 197:1092-1094 (1977); Bayer, Exp. Brain Res., 46: 315-3323 (1982); Altman et al., J. Comp. Neurol., 301:365-381 (1990)). Neurons are born during a narrow window of time and, when differentiated, become blocked at the early G1(G0) phase of the cell cycle (Angevine et al., Nature, 192:766-768 (1961); Takahashi et al., J. Neurocytol., 21:185-197 (1992)). Accordingly, differentiated neurons are not capable of proceeding from the G1(G0) phase to the DNA synthesis phase. Thus, differentiated neurons are not only incapable of synthesizing DNA, but also are incapable of proceeding through the rest of the cell cycle to form new cells (proliferating).
It would be advantageous if one could cause normally differentiated neurons to proceed from the G1(G0) phase to the DNA synthesis phase. By doing so, one could induce proliferation of such neurons to produce new cells.
Moreover, as demonstrated by the present inventors, one can use gene therapy by stably integrating DNA into normally differentiated neurons that have been induced to proceed from the G1(G0) phase to the DNA synthesis phase. Gene therapy involves incorporating nucleic acid into the patient host. In certain applications, the host will express the foreign nucleic acid such that a therapeutic protein is made in the host. Although gene therapy has been used to express nucleic acids in nondifferentiated cells that can proliferate and synthesize nucleic acid, unique issues exist for gene therapy involving transfer of nucleic acid into neurons.
For gene therapy, one should consider factors involved in the delivery of the nucleic acid into the target cell and efficient expression of the nucleic acid in the cell. For therapy involving neurons as the target cells, customarily, one would transfer the nucleic acid in vivo to postmitotic (nonproliferating and fully differentiated) cells. Therapy for diseases of or trauma to the central nervous system may involve use of differentiated neurons as the target cells.
Physical and viral vector methods have been used for gene transfer into neurons of the adult nervous system with limited success. Direct injection of DNA into neurons is possible, however, this is limiting because of the number of cells involved.
Although liposomes, complex ligand DNA conjugates, or viral vectors can deliver DNA to differentiated cells, the subsequent expression of DNA is transient since such cells do not normally synthesize DNA. Gene therapy would be somewhat limited if only transient expression of the transferred DNA was achieved rather than stable functional integration of the transferred DNA because of the shorter periods of expression associated with transient expression.
For many conditions, one desires expression of the DNA over an extended period of time to provide the therapeutic molecule for ongoing treatment. For example, a patient having Parkinson's disease or Alzheimers disease often needs treatment for many years. Functional integration of transferred DNA would provide such long term expression of the therapeutic molecule. Thus, it would be desirable to have stable functional integration of the transferred DNA into neurons for treatment of neurodegenerative diseases and trauma.
The regulation of G1(G0) to DNA synthesis transition involves regulator molecules known as transcription factors. Modification (mainly phosphorylation) of preexisting regulators and transcriptional activation of new genes occurs during this process. Several different protein kinases which form complexes and modify transcription factors are described (Devoto et al., Cell, 68:167-176 (1992), Hunter, Cell, 75:839-841 (1993)). Several transcription factors are described which regulate different steps in G1 to DNA synthesis transition. Retinoblastoma antigen (Rb) (Hamel et al., Molec. Cell. Biol., 12:3431-3438 (1992)), sequence specific transcription factor E2F (Nevins, Science, 258:424-429 (1992), and tumor suppressor p53 (Zambetti et al., Genes and Development, 6:1143-1152 (1992)) play central roles in the initiation of DNA synthesis and the DNA synthesis phase.
Transcription factor E2F is a key molecule in the G1 to DNA synthesis phase transition. E2F regulates expression of genes necessary for DNA synthesis phase initiation and progression. Nevins, Science, 258:424-429 (1992); Mudryj et al, Cell, 65:1243-1253 (1991); Schwarz et al., EMBO J., 12:1013-1020 (1993). During the early G1 phase, the cellular E2F is in a complex with Rb (Nevins, 1992). In this complex, the E2F is inactive. Also, the Rb/E2F complex is an active repressor of transcription of several S phase genes (Weintraub et al., Nature, 358:259-261 (1992)).
Phosphorylation of Rb by cdc2 or cdk kinases releases E2F, which activates the genes necessary in the DNA synthesis phase. Also, adenovirus oncoprotein E1A is a transcriptional regulator that binds Rb, which causes the release of active E2F from the inactive complex with Rb.
During the DNA synthesis phase, the E2F will become inactivated again by forming a complex with cyclin A, cdc2 kinase and p107 (Pagano et al., Science, 255:1144-1147 (1992)). Adenovirus oncoprotein E1A also binds p107, which causes release of active E2F from the inactive complexes with p107. Complexes of E1A with Rb and p107 are more stable than E2F complexes with these same proteins; thus, the presence of E1A causes dissociation of E2F complexes with Rb and p107 and the release of transcriptionally active E2F (Nevins, Science, 258:424-429 (1992)).
Different forms of E2F exist in different cell types (Kaelin et al., Cell, 70:351-364 (1992). The homology between cell cycle regulatory factors from different species (e.g., from yeast to humans), however, is very high. Accordingly, one can accurately predict the action of such factors in the cell cycle regulation from one species to another.
Although the present inventors do not intend to be limited to any theory of why differentiated neurons do not synthesize DNA, since mature neurons in cerebral cortex and cerebellum express Rb (Okano et al., J. Neurosci., 13:2930-2938 (1993) and Bernards et al., Proc. Nat. Acad. Sci. U.S.A., 86:6474-6478 (1989)) and E2F (Helin et al, Cell, 70:337-350 (1992)) at relatively high levels, one hypothesis of the inventors is that the cell cycle is blocked by formation of inactive complexes between Rb and E2F in these neurons. Alternatively, Rb may have an important role in differentiation and functioning of neurons.
An object of the present invention is to provide methods of inducing DNA synthesis in normally differentiated cells such as neurons. Another object according to certain embodiments of the present invention is to induce normally differentiated cells such as neurons to proliferate.
The present inventors have shown that transfecting neurons with DNA encoding transcription factor E2F1 and viral oncoprotein E1A in vitro and in vivo results in the induction of DNA synthesis in these neurons. The present inventors have also shown that such transfection in vivo, followed by induction of DNA synthesis, can be applied to stably integrate a functional gene(s) that is also transfected into the neuron.
By inducing differentiated neurons to synthesize DNA, the present invention provides for production of protein in neurons. With stable and functional integration, the present invention also provides for gene therapy in which normally differentiated neurons are induced to produce a given therapeutic protein. Also, by commencing the cell cycle from the G1(G0) phase to DNA synthesis, the present invention is also directed to inducing proliferation of neurons to produce new cells.
Another object of the present invention is to provide a method for isolating DNA that encodes molecules that regulate or induce DNA synthesis or proliferation of differentiated cells such as neurons. The method includes (a) preparing a subtraction cDNA library, (b) cloning individual cDNAs into individual vectors to create an expression library, transfecting differentiated cells with the expression library, (c) selecting transfected cells that synthesize DNA or proliferate, (d) isolating cDNAs from the selected cells, and (e) selecting cDNAs other than cDNAs known to encode molecules that regulate or induce cells progressing to the DNA synthesis phase of the cell cycle.
The method according to certain embodiments further includes the step of amplifying isolated cDNAs from steps (d) and (e) in a polymerase chain reaction. The amplified cDNAs may then be cloned into an expression vector which is transfected into differentiated cells such as neurons to induce DNA synthesis and/or proliferation.
DNA isolated when using such methods are also provided.
Another object according to certain embodiments of the invention is to use DNA encoding molecules that induce differentiated cells to synthesize DNA to isolate homologous genes from other organisms including humans, to produce proteins and antibodies against proteins for which these genes code, and to induce differentiated cells such as neurons to proliferate. The antibodies can be used for diagnostic purposes or for treatment of conditions such as cancer where the patient already produces too much of the molecule that induces DNA synthesis.
Another object according to certain embodiments of the present invention is that it enables one to use neurons from the same individual to obtain (induce the production of) more neurons. Thus, it is possible to replace lost neurons after injury or neurodegenerative diseases.
Another object according to certain embodiments of the present invention is to induce differentiated cells such as neurons to produce proteins in vitro or in vivo by inducing DNA synthesis in such differentiated cells.
Other objects and advantages will become apparent upon review of this patent application.